fnbB DNA from CUDC-907 strains 8325-4, N315, MSSA476 and P1 was used as control. Identification of novel FnBPB isotypes (Types V, VI and VII) The fnbB gene fragments amplified from S. aureus strains 2 (ST7) 114 (ST39), 233 (ST45), 304 (ST39), CP-690550 clinical trial 138 (ST30), 563 (ST37), 3077 (ST17) and 3110 (ST12) did not hybridise to probes specific for FnBPB isotypes I-IV. The fnbB gene fragments from these strains were cloned and sequenced, and the deduced A domain amino acid sequences were compared to the sequences of A domains of types I – IV. S. aureus strains 2 (ST7)

and 3110 (ST12) specify a novel FnBPB A domain called isotype V (N23, 68.8 – 73.3% identical to isotypes I – IV). The A domains of strains 3077 (ST17) and 233 (ST45) are also different and are called isotype VI (N23, 66.0- 76.6% identical to types I – V) and isotype VII (N23, 66.2% – 85% identical to types I-VI) (Table 1). Strains TH-302 in vitro 114, 563, 138 and 304 specify an identical

A domain which is 92% identical to isotype II and is called isotype II* (Table 1) Phylogenetic analysis of FnBPB A domain isotypes I-VII Figure 3 shows a neighbour-joining phylogenetic tree which was constructed based upon the concatenated sequences of the seven housekeeping genes used for MLST analysis. As MLST reflects the evolution of the stable core genome [23], this tree describes the phylogenetic relatedness of the S. aureus strains studied here. It is separated into two major clusters as was also shown previously in a detailed phylogenetic analysis of thirty diverse S.aureus isolates [24]. The FnBPB A domain isotypes specified by each genotype (as predicted by DNA hybridisation or sequencing) are indicated. The phylogeny of fnbB alleles illustrated here does not correspond to that of the core genome as determined by MLST. For example, two strains that cluster together in Group 1 (ST49 and ST52) carry fnbB genes encoding isotype II, as do distantly related strains from Group 2 (ST5 and ST18).

Conversely, clustered strains such as ST8 and ST97 from Group 2 contain fnbB genes encoding isotypes I and IV, respectively. Isolates belonging to the Docetaxel manufacturer same ST (ST45) were found to specify different FnBPB isotypes (II and VII). These results suggest that fnbB alleles have dispersed by horizontal transfer, most likely by homologous recombination. Figure 3 Neighbour-joining tree based upon concatenated sequences of MLST alleles from human S. aureus strains. MLST allele sequences representing each clinical strain studied here were used to generate a neighbour joining tree using MEGA 4. The A domain isotypes carried by strains of each MLST genotype, determined by sequencing and hybridization analysis, are indicated. The dashed line indicates the separation of the MLST genotypes into Groups 1 and 2, which is based on sequence data from MLST alleles and other unlinked loci [24].